Cellular Heterogeneity in Adipose Tissues.

Adipose tissue depots in distinct anatomical locations mediate key aspects of metabolism, including energy storage, nutrient release, and thermogenesis. Although adipocytes make up more than 90% of adipose tissue volume, they represent less than 50% of its cellular content. Here, I review recent advances in genetic lineage tracing and transcriptomics that reveal the identities of the heterogeneous cell populations constituting mouse and human adipose tissues. In addition to mature adipocytes and their progenitors, these include endothelial and various immune cell types that together orchestrate adipose tissue development and functions. One salient finding is the identification of progenitor subtypes that can modulate adipogenic capacity through paracrine mechanisms. Another is the description of fate trajectories of monocyte/macrophages, which can respond maladaptively to nutritional and thermogenic stimuli, leading to metabolic disease. These studies have generated an extraordinary source of publicly available data that can be leveraged to explore commonalities and differences among experimental models, providing new insights into adipose tissues and their role in metabolic disease.

Angiogenesis in adipose tissue and obesity.

While most tissues exhibit their greatest growth during development, adipose tissue is capable of additional massive expansion in adults. Adipose tissue expandability is advantageous when temporarily storing fuel for use during fasting, but becomes pathological upon continuous food intake, leading to obesity and its many comorbidities. The dense vasculature of adipose tissue provides necessary oxygen and nutrients, and supports delivery of fuel to and from adipocytes under fed or fasting conditions. Moreover, the vasculature of adipose tissue comprises a major niche for multipotent progenitor cells, which give rise to new adipocytes and are necessary for tissue repair. Given the multiple, pivotal roles of the adipose tissue vasculature, impairments in angiogenic capacity may underlie obesity-associated diseases such as diabetes and cardiometabolic disease. Exciting new studies on the single-cell and single-nuclei composition of adipose tissues in mouse and humans are providing new insights into mechanisms of adipose tissue angiogenesis. Moreover, new modes of intercellular communication involving micro vesicle and exosome transfer of proteins, nucleic acids and organelles are also being recognized to play key roles. This review focuses on new insights on the cellular and signaling mechanisms underlying adipose tissue angiogenesis, and on their impact on obesity and its pathophysiological consequences.

Diverse repertoire of human adipocyte subtypes develops from transcriptionally distinct mesenchymal progenitor cells.

Single-cell sequencing technologies have revealed an unexpectedly broad repertoire of cells required to mediate complex functions in multicellular organisms. Despite the multiple roles of adipose tissue in maintaining systemic metabolic homeostasis, adipocytes are thought to be largely homogenous with only 2 major subtypes recognized in humans so far. Here we report the existence and characteristics of 4 distinct human adipocyte subtypes, and of their respective mesenchymal progenitors. The phenotypes of these distinct adipocyte subtypes are differentially associated with key adipose tissue functions, including thermogenesis, lipid storage, and adipokine secretion. The transcriptomic signature of "brite/beige" thermogenic adipocytes reveals mechanisms for iron accumulation and protection from oxidative stress, necessary for mitochondrial biogenesis and respiration upon activation. Importantly, this signature is enriched in human supraclavicular adipose tissue, confirming that these cells comprise thermogenic depots in vivo, and explain previous findings of a rate-limiting role of iron in adipose tissue browning. The mesenchymal progenitors that give rise to beige/brite adipocytes express a unique set of cytokines and transcriptional regulators involved in immune cell modulation of adipose tissue browning. Unexpectedly, we also find adipocyte subtypes specialized for high-level expression of the adipokines adiponectin or leptin, associated with distinct transcription factors previously implicated in adipocyte differentiation. The finding of a broad adipocyte repertoire derived from a distinct set of mesenchymal progenitors, and of the transcriptional regulators that can control their development, provides a framework for understanding human adipose tissue function and role in metabolic disease.

Visualization of self-delivering hydrophobically modified siRNA cellular internalization.

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.

Exercise Rescues Gene Pathways Involved in Vascular Expansion and Promotes Functional Angiogenesis in Subcutaneous White Adipose Tissue.

Exercise mitigates chronic diseases such as diabetes, cardiovascular diseases, and obesity; however, the molecular mechanisms governing protection from these diseases are not completely understood. Here we demonstrate that exercise rescues metabolically compromised high fat diet (HFD) fed mice, and reprograms subcutaneous white adipose tissue (scWAT). Using transcriptomic profiling, scWAT was analyzed for HFD gene expression changes that were rescued by exercise. Gene networks involved in vascularization were identified as prominent targets of exercise, which led us to investigate the vasculature architecture and endothelial phenotype. Vascular density in scWAT was found to be compromised in HFD, and exercise rescued this defect. Similarly, angiogenic capacity as measured by ex vivo capillary sprouting was significantly promoted with exercise. Together, these data demonstrate that exercise enhances scWAT vascularization and functional capacity for angiogenesis, and can prevent the detrimental effects of HFD. The improvement in these indices correlates with improvement of whole-body metabolism, suggesting that scWAT vascularization may be a potential therapeutic target for metabolic disease.

Human White Adipocytes Convert Into "Rainbow" Adipocytes In Vitro.

White adipocytes are plastic cells able to reversibly transdifferentiate into brown adipocytes and into epithelial glandular cells under physiologic stimuli in vivo. These plastic properties could be used in future for regenerative medicine, but are incompletely explored in their details. Here, we focused on plastic properties of human mature adipocytes (MA) combining gene expression profile through microarray analysis with morphologic data obtained by electron and time lapse microscopy. Primary MA showed the classic morphology and gene expression profile of functional mature adipocytes. Notably, despite their committed status, MA expressed high levels of reprogramming genes. MA from ceiling cultures underwent transdifferentiation toward fibroblast-like cells with a well-differentiated morphology and maintaining stem cell gene signatures. The main morphologic aspect of the transdifferentiation process was the secretion of large lipid droplets and the development of organelles necessary for exocrine secretion further supported the liposecretion process. Of note, electron microscope findings suggesting liposecretion phenomena were found also in explants of human fat and rarely in vivo in fat biopsies from obese patients. In conclusion, both MA and post-liposecretion adipocytes show a well-differentiated phenotype with stem cell properties in line with the extraordinary plasticity of adipocytes in vivo. J. Cell. Physiol. 232: 2887-2899, 2017. © 2016 Wiley Periodicals, Inc.

Human adipose tissue expansion in pregnancy is impaired in gestational diabetes mellitus.

AIMS/HYPOTHESIS: During pregnancy, adipose tissue (AT) must expand to support the growing fetus and the future nutritional needs of the offspring. Limited expandability of AT is associated with insulin resistance, attributed to ectopic lipid deposition. This study aimed to investigate human AT expandability during pregnancy and its role in the pathogenesis of gestational diabetes mellitus (GDM). METHODS: This cross-sectional study of omental (OM) and subcutaneous (SQ) AT collected at Caesarean delivery included 11 pregnant and three non-pregnant women with normal glucose tolerance (NGT), five with GDM, three with type 2 diabetes mellitus. Adipocyte size, capillary density, collagen content and capillary growth were measured. Affymetrix arrays and real-time PCR studies of gene expression were performed. RESULTS: Mean OM adipocyte size was greater in women with GDM than in those with NGT (p = 0.004). Mean OM and SQ capillary density was lower in GDM compared with NGT (p = 0.015). Capillary growth did not differ significantly between groups. The most differentially expressed AT transcript when comparing non-pregnant and pregnant women corresponded to the IGF binding protein (IGFBP)-5, the expression levels of which was found by subsequent quantitative real-time PCR to be lower in women with GDM vs women with NGT (p < 0.0001). CONCLUSIONS/INTERPRETATION: The relative OM adipocyte hypertrophy and decreased OM and SQ capillary density are consistent with impaired AT expandability in GDM. The induction of adipose tissue IGFBP5 in pregnancy and its decrease in GDM point to the importance of the IGF-1 signalling pathway in AT expansion in pregnancy and GDM susceptibility.

Control of adipose tissue expandability in response to high fat diet by the insulin-like growth factor-binding protein-4.

Adipose tissue expansion requires growth and proliferation of adipocytes and the concomitant expansion of their stromovascular network. We have used an ex vivo angiogenesis assay to study the mechanisms involved in adipose tissue expansion. In this assay, adipose tissue fragments placed under pro-angiogenic conditions form sprouts composed of endothelial, perivascular, and other proliferative cells. We find that sprouting was directly stimulated by insulin and was enhanced by prior treatment of mice with the insulin sensitizer rosiglitazone. Moreover, basal and insulin-stimulated sprouting increased progressively over 30 weeks of high fat diet feeding, correlating with tissue expansion during this period. cDNA microarrays analyzed to identify genes correlating with insulin-stimulated sprouting surprisingly revealed only four positively correlating (Fads3, Tmsb10, Depdc6, and Rasl12) and four negatively correlating (Asph, IGFbp4, Ppm1b, and Adcyap1r1) genes. Among the proteins encoded by these genes, IGFbp4, which suppresses IGF-1 signaling, has been previously implicated in angiogenesis, suggesting a role for IGF-1 in adipose tissue expandability. Indeed, IGF-1 potently stimulated sprouting, and the presence of activated IGF-1 receptors in the vasculature was revealed by immunostaining. Recombinant IGFbp4 blocked the effects of insulin and IGF-1 on mouse adipose tissue sprouting and also suppressed sprouting from human subcutaneous adipose tissue. These results reveal an important role of IGF-1/IGFbp4 signaling in post-developmental adipose tissue expansion.

Adipose tissue angiogenesis: impact on obesity and type-2 diabetes.

The growth and function of tissues are critically dependent on their vascularization. Adipose tissue is capable of expanding many-fold during adulthood, therefore requiring the formation of new vasculature to supply growing and proliferating adipocytes. The expansion of the vasculature in adipose tissue occurs through angiogenesis, where new blood vessels develop from those pre-existing within the tissue. Inappropriate angiogenesis may underlie adipose tissue dysfunction in obesity, which in turn increases type-2 diabetes risk. In addition, genetic and developmental factors involved in vascular patterning may define the size and expandability of diverse adipose tissue depots, which are also associated with type-2 diabetes risk. Moreover, the adipose tissue vasculature appears to be the niche for pre-adipocyte precursors, and factors that affect angiogenesis may directly impact the generation of new adipocytes. Here we review recent advances on the basic mechanisms of angiogenesis, and on the role of angiogenesis in adipose tissue development and obesity. A substantial amount of data points to a deficit in adipose tissue angiogenesis as a contributing factor to insulin resistance and metabolic disease in obesity. These emerging findings support the concept of the adipose tissue vasculature as a source of new targets for metabolic disease therapies. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

Understanding multifactorial influences on the continuum of maternal weight trajectories in pregnancy and early postpartum: study protocol, and participant baseline characteristics.

BACKGROUND: Maternal and offspring immediate and long-term health are affected by pregnancy weight gain and maternal weight. This study was designed to determine feasibility of: 1) recruiting a socio-economically and racially/ethnically diverse sample of pregnant women into a longitudinal observational study, including consenting the women for serial biologic specimen evaluations; 2) implementing comprehensive assessments (including biologic, anthropometric, behavioral, cognitive/psychosocial and socio-demographic, and cultural measures) at multiple time points over the study period, including collecting biologic specimens at planned and unplanned pregnancy delivery times; and 3) retaining the sample for one year into the postpartum period. Additionally, the study will provide preliminary data of associations among hypothesized predictors, mediators and moderators of pregnancy and post-partum maternal and infant weight trajectories. The study was conceptualized under a Biopsychosocial Model using a lifespan approach. Study protocol and baseline characteristics are described. METHODS/DESIGN: We sought to recruit a sample of 100 healthy women age 18-45 years, between 28-34 weeks gestation, with singleton pregnancies, enrolled in care prior to 17 weeks gestation. Women provide written consent for face-to-face (medical history, anthropometrics, biologic specimens), and paper-and-pencil assessments, at five time points: baseline (third trimester), delivery-associated, and 6-weeks, 3-months and 6-months postpartum. Additional telephone-based assessments (diet, physical activity and breastfeeding) administered baseline and three-months postpartum. Infant weights are collected until 1-year of life. We seek to retain 80% of participants at six-months postpartum and 80% of offspring at 12-months. 110 women were recruited. Sample characteristics include: mean age 28.3 years, BMI 25.7 kg/m(2), and gestational age at baseline visit of 32.5 weeks. One-third of cohort was non-white, over a quarter were Latina, and almost a quarter were non-US born. The cohort majority was multigravida, had graduated high school and/or had higher levels of education, and worked outside the home. DISCUSSION: Documentation of study feasibility and preliminary data for theory-driven hypothesis of maternal and child factors associated with weight trajectories will support future large scale longitudinal studies of risk and protective factors for maternal and child health. This research will also inform intervention targets facilitating healthy maternal and child weight.

Rabenosyn-5 defines the fate of the transferrin receptor following clathrin-mediated endocytosis.

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.

Adipose tissue as a linchpin of organismal ageing.

Ageing is a conserved biological process, modulated by intrinsic and extrinsic factors, that leads to changes in life expectancy. In humans, ageing is characterized by greatly increased prevalence of cardiometabolic disease, type 2 diabetes and disorders associated with impaired immune surveillance. Adipose tissue displays species-conserved, temporal changes with ageing, including redistribution from peripheral to central depots, loss of thermogenic capacity and expansion within the bone marrow. Adipose tissue is localized to discrete depots, and also diffusely distributed within multiple organs and tissues in direct proximity to specialized cells. Thus, through their potent endocrine properties, adipocytes are capable of modulating tissue and organ function throughout the body. In addition to adipocytes, multipotent progenitor/stem cells in adipose tissue play a crucial role in maintenance and repair of tissues throughout the lifetime. Adipose tissue may therefore be a central driver for organismal ageing and age-associated diseases. Here we review the features of adipose tissue during ageing, and discuss potential mechanisms by which these changes affect whole-body metabolism, immunity and longevity. We also explore the potential of adipose tissue-targeted therapies to ameliorate age-associated disease burdens.

linc-ADAIN, a human adipose lincRNA, regulates adipogenesis by modulating KLF5 and IL-8 mRNA stability.

Adipose tissue remodeling and dysfunction, characterized by elevated inflammation and insulin resistance, play a central role in obesity-related development of type 2 diabetes (T2D) and cardiovascular diseases. Long intergenic non-coding RNAs (lincRNAs) are important regulators of cellular functions. Here, we describe the functions of linc-ADAIN (adipose anti-inflammatory), an adipose lincRNA that is downregulated in white adipose tissue of obese humans. We demonstrate that linc-ADAIN knockdown (KD) increases KLF5 and interleukin-8 (IL-8) mRNA stability and translation by interacting with IGF2BP2. Upregulation of KLF5 and IL-8, via linc-ADAIN KD, leads to an enhanced adipogenic program and adipose tissue inflammation, mirroring the obese state, in vitro and in vivo. KD of linc-ADAIN in human adipose stromal cell (ASC) hTERT adipocytes implanted into mice increases adipocyte size and macrophage infiltration compared to implanted control adipocytes, mimicking hallmark features of obesity-induced adipose tissue remodeling. linc-ADAIN is an anti-inflammatory lincRNA that limits adipose tissue expansion and lipid storage.

Structural basis for Rab GTPase recognition and endosome tethering by the C2H2 zinc finger of Early Endosomal Autoantigen 1 (EEA1).

Regulation of endosomal trafficking by Rab GTPases depends on selective interactions with multivalent effectors, including EEA1 and Rabenosyn-5, which facilitate endosome tethering, sorting, and fusion. Both EEA1 and Rabenosyn-5 contain a distinctive N-terminal C(2)H(2) zinc finger that binds Rab5. How these C(2)H(2) zinc fingers recognize Rab GTPases remains unknown. Here, we report the crystal structure of Rab5A in complex with the EEA1 C(2)H(2) zinc finger. The binding interface involves all elements of the zinc finger as well as a short N-terminal extension but is restricted to the switch and interswitch regions of Rab5. High selectivity for Rab5 and, to a lesser extent Rab22, is observed in quantitative profiles of binding to Rab family GTPases. Although critical determinants are identified in both switch regions, Rab4-to-Rab5 conversion-of-specificity mutants reveal an essential requirement for additional substitutions in the proximal protein core that are predicted to indirectly influence recognition through affects on the structure and conformational stability of the switch regions.

Regulation of lipolysis by 14-3-3 proteins on human adipocyte lipid droplets.

Adipocyte lipid droplets (LDs) play a crucial role in systemic lipid metabolism by storing and releasing lipids to meet the organism's energy needs. Hormonal signals such as catecholamines and insulin act on adipocyte LDs, and impaired responsiveness to these signals can lead to uncontrolled lipolysis, lipotoxicity, and metabolic disease. To investigate the mechanisms that control LD function in human adipocytes, we applied proximity labeling mediated by enhanced ascorbate peroxidase (APEX2) to identify the interactome of PLIN1 in adipocytes differentiated from human mesenchymal progenitor cells. We identified 70 proteins that interact specifically with PLIN1, including PNPLA2 and LIPE, which are the primary effectors of regulated triglyceride hydrolysis, and 4 members of the 14-3-3 protein family (YWHAB, YWHAE, YWHAZ, and YWHAG), which are known to regulate diverse signaling pathways. Functional studies showed that YWHAB is required for maximum cyclic adenosine monophosphate (cAMP)-stimulated lipolysis, as its CRISPR-Cas9-mediated knockout mitigates lipolysis through a mechanism independent of insulin signaling. These findings reveal a new regulatory mechanism operating in human adipocytes that can impact lipolysis and potentially systemic metabolism.

Wnt signaling preserves progenitor cell multipotency during adipose tissue development.

Mesenchymal stem/progenitor cells are essential for tissue development and repair throughout life, but how they are maintained under chronic differentiation pressure is not known. Using single-cell transcriptomics of human progenitor cells we find that adipose differentiation stimuli elicit two cellular trajectories: one toward mature adipocytes and another toward a pool of non-differentiated cells that maintain progenitor characteristics. These cells are induced by transient Wnt pathway activation and express numerous extracellular matrix genes and are therefore named structural Wnt-regulated adipose tissue cells. We find that the genetic signature of structural Wnt-regulated adipose tissue cells is present in adult human adipose tissue and adipose tissue developed from human progenitor cells in mice. Our results suggest a mechanism whereby adipose differentiation occurs concurrently with the maintenance of a mesenchymal progenitor cell pool, ensuring tissue development, repair and appropriate metabolic control over the lifetime.

A distinct class of hematopoietic stem cells develop from the human yellow bone marrow.

Aging and metabolic diseases are accompanied by systemic inflammation, but the mechanisms that induce this state are not known. We developed a human bone-marrow organoid system to explore mechanisms underlying metabolic-disease associated systemic inflammation. We find that a distinct type of hematopoietic stem cell (HSC) develops in the adipose-rich, yellow bone marrow, which is known to gradually replace the hematopoietic red marrow as we age and during metabolic disease. Unlike HSCs derived from the red bone marrow, HSCs derived from the yellow bone marrow have higher proliferation rates, increase myeloid differentiation, skew towards pro-inflammatory M1 macrophage differentiation, and express a distinct transcriptomic profile associated with responsiveness to wounding. Yellow marrow-derived HSCs express higher levels of the leptin receptor, which we find to be further increased in patients with type 2 diabetes. Our work demonstrates that the human long bone yellow marrow is a niche for a distinct class of HSCs which could underlie hematopoietic dysfunction during aging and metabolic disease processes suggesting a shared inflammaging mechanism.

Adenylate kinase 2 links mitochondrial energy metabolism to the induction of the unfolded protein response.

The unfolded protein response (UPR) is a homeostatic signaling mechanism that balances the protein folding capacity of the endoplasmic reticulum (ER) with the secretory protein load of the cell. ER protein folding capacity is dependent on the abundance of chaperones, which is increased in response to UPR signaling, and on a sufficient ATP supply for their activity. An essential branch of the UPR entails the splicing of XBP1 mRNA to form the XBP1 transcription factor. XBP1 has been shown to be required during adipocyte differentiation, enabling mature adipocytes to secrete adiponectin, and during differentiation of B cells into antibody-secreting plasma cells. Here we find that adenylate kinase 2 (AK2), a mitochondrial enzyme that regulates adenine nucleotide interconversion within the intermembrane space, is markedly induced during adipocyte and B cell differentiation. Depletion of AK2 by RNAi impairs adiponectin secretion in 3T3-L1 adipocytes, IgM secretion in BCL1 cells, and the induction of the UPR during differentiation of both cell types. These results reveal a new mechanism by which mitochondria support ER function and suggest that specific mitochondrial defects may give rise to impaired UPR signaling. The requirement for AK2 for UPR induction may explain the pathogenesis of the profound hematopoietic defects of reticular dysgenesis, a disease associated with mutations of the AK2 gene in humans.

Depot-specific differences and insufficient subcutaneous adipose tissue angiogenesis in human obesity.

BACKGROUND: Adipose tissue expands in response to excess caloric intake, but individuals prone to deposit visceral instead of subcutaneous adipose tissue have higher risk of metabolic disease. The role of angiogenesis in the expandability of human adipose tissue depots is unknown. The objective of this study was to measure angiogenesis in visceral and subcutaneous adipose tissue and to establish whether there is a relationship between obesity, metabolic status, and the angiogenic properties of these depots. METHODS AND RESULTS: Angiogenic capacity was determined by quantifying capillary branch formation from human adipose tissue explants embedded in Matrigel, and capillary density was assessed by immunohistochemistry. Subcutaneous adipose tissue had a greater angiogenic capacity than visceral tissue, even after normalization to its higher initial capillary density. Gene array analyses revealed significant differences in expression of angiogenic genes between depots, including an increased subcutaneous expression of angiopoietin-like protein 4, which is proangiogenic in an adipose tissue context. Subcutaneous capillary density and angiogenic capacity decreased with morbid obesity, and subcutaneous, but not visceral, adipose tissue angiogenic capacity correlated negatively with insulin sensitivity. CONCLUSIONS: These data imply that subcutaneous adipose tissue has a higher capacity to expand its capillary network than visceral tissue, but this capacity decreases with morbid obesity. The decrease correlates with insulin resistance, suggesting that impairment of subcutaneous adipose tissue angiogenesis may contribute to metabolic disease pathogenesis.